Molecular characterization of a novel victorivirus isolated from the phytopathogenic fungus Phaeobotryon rhois

Phaeobotryon rhois is an important pathogenic fungus that causes dieback and canker disease of woody hosts. A novel mycovirus, tentatively named "Phaeobotryon rhois victorivirus 1" (PrVV1), was identified in P. rhois strain SX8-4. The PrVV1 has a double-stranded RNA (dsRNA) genome that is 5,224 base pairs long and contains two open reading frames (ORF1 and ORF2), which overlap at a AUGA sequence. ORF1 encodes a polypeptide of 786 amino acids (aa) that contains the conserved coat protein (CP) domain of victoriviruses, while ORF2, encodes a large polypeptide of 826 aa that contains the conserved RNA-dependent RNA polymerase (RdRp) domain of victoriviruses. Our analysis of genomic structure, homology, and phylogeny indicated that PrVV1 is a novel member of the genus Victorivirus in the family Totiviridae. This is the first report of the complete genome sequence of a victorivirus that infects P. rhois.

Phaeobotryon rhois, belonging to the family Botryosphaeriaceae, is an important pathogenic fungus that causes dieback and canker disease in woody hosts [15,16]. Interestingly, mycoviruses have been reported in many types of fungi that cause these types of diseases [7,17,18], but none have been reported in P. rhois.
Here, we report a novel dsRNA mycovirus isolated from P. rhois strain SX8-4, tentatively named "Phaeobotryon rhois victorivirus 1" (PrVV1). Genomic structure analysis and phylogenetic analysis showed that PrVV1 is closely related to members of the genus Victorivirus. To the best of our knowledge, this is the first report of a mycovirus of the family Totiviridae that infects P. rhois. Provenance and sequencing of strains P. rhois strain SX8-4 was isolated from the tips of Ziziphus jujuba collected in Shanxi province, China. For RNA extraction, strain SX8-4 was cultured on a potato dextrose agar (PDA) plate overlaid with a cellophane membrane for five days at 28ºC (Fig. 1a), and the mycelia were harvested and ground to a fine powder in liquid nitrogen using the CF-11 cellulose powder method described by Zhao et al. [19]. The extracted nucleic acids were digested with DNase I and S1 nuclease to eliminate DNA and ssRNA. The purified dsRNA was separated by electrophoresis in a 1.2% gel in 1× TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.1) at 130 V and imaged using a gel documentation system. A cDNA library of P. rhois strain SX8-4 dsRNA was constructed using a reverse transcription kit (TransGen Biotech), following the manufacturer's protocols. For sequencing, the specific dsRNA band was excised from the gel, purified, and subjected to random amplification by RT-PCR. The resulting cDNA was cloned using a random primer (5'-CGA TCG ATC ATG ATG CAA TGC-NNNNNN-3') amplification method and then amplified using a single specific primer (5'-CGA TCG ATC ATG ATG CAA TGC-3'). Sequencing and analysis of the cDNA sequences were carried out as described previously [19]. Sequence gaps were filled by RT-PCR using specific primers designed based on the sequences obtained, and the terminal sequences were completed by ligase-mediated rapid amplification of cDNA ends [20]. All of the amplified PCR products were cloned into the vector pMD18-T and introduced into E. coli DH5α cells using the heat shock transformation method. Clones containing inserts were sequenced at TsingKe Biological Technology Co. Ltd. (Beijing, China).
The resulting sequences were assembled using DNA-MAN software and analyzed using Bioedit software, ORFs were predicted using ORFfinder at the National Center for Biotechnology Information (NCBI) website. Potential secondary structures in the 5′-and 3′-UTRs of PrVV1 were predicted using Mfold version 2.3. Phylogenetic analysis was performed using MEGA 11 software [21]. All sequences used as references were obtained from the NCBI database.

Sequence properties
Sequence analysis showed that P. rhois strain SX8-4 contained a dsRNA virus (PrVV1) belonging to the genus of Victorivirus in the family Totiviridae. Agarose gel electrophoresis revealed a single segment about 5,000 bp in size (Fig. 1b). The full genome sequence of PrVV1 was determined and found to be 5,224 bp in length, containing 17.3% A, 35.5% C, 29.9% G, and 17.3% U. It contains two ORFs, as shown in Fig. 1c. ORF1 is 2,361 nt long (nt positions 298 to 2658) and encodes a 786-aa protein with a molecular weight of 81.6 kDa. Based on predictive analysis using the CDsearch program on the NCBI website, ORF1 encodes a conserved coat protein domain at nt positions 340 to 2421. ORF2 is 2481 nt long (nt positions 2,655 to 5,135) and encodes an 826-aa protein with a molecular weight of 91.1 kDa. ORF2 encodes a conserved RdRp domain at nt positions 2,955 to 4,376 (Fig. 1c). The tetranucleotide AUGA at nt positions 2,655 to 2,658 is a motif that allows the translation of the downstream ORF2 by a termination-reinitiation mechanism at the overlapping ORF1 stop codon and ORF2 start codon (Fig. 1c). The AUGA tetranucleotide is a common feature of victoriviruses [11,13,14]. In addition, an H-type pseudoknot structure (GAA ggagccgCggccGCU GCA ggcc cggcuccCCA ACA AUGA ) was predicted upstream of the AUGA motif. It has almost perfect reverse complementarity between the sequences "ggagccgCggcc" and "ggcc cggcucc" (except for one "C" base gap) and is therefore likely to form a stem-loop structure. The 5' untranslated region (UTR) and the 3' UTR are 297 nt (nt positions 1 to 297) and 89 nt (nt positions 5,136 to 5,224) long, respectively. Both regions are predicted to have stable secondary structures (Fig. 1d). The genome sequence of PrVV1 has been submitted to the GenBank database under accession number OP100309.
A multiple aa sequence alignment of the RdRps of PrVV1 and some members of the family Totiviridae revealed several conserved motifs (Fig. 2a). A phylogenetic tree based on these sequences was constructed using the maximum-likelihood method (Fig. 2b) and showed that PrVV1 clustered with three mycoviruses (MpVV2, DsVV1, and NpVV2) to form a clade, which, together with two other clades, constituted a larger clade representing the members of the genus Victorivirus. Another phylogenetic tree, based on CP sequences, was also constructed, and it showed a very similar topology (Fig. 2c). PrVV1 is therefore a novel member of the genus Victorivirus, family Totiviridae.
In conclusion, based on sequence comparisons, genome organization, ORF characterization, and phylogenetic analysis, PrVV1 should be considered as a new member of the genus Victorivirus in the family Totiviridae. To the best of our knowledge, this is not only the first report of a victorivirus infecting P. rhois but also the first report of any mycovirus in P. rhois.